Evaluation of pretreatment methods for filamentous fungal detection

In order to obtain the best mass spectrometry identification results for using the most appropriate methods in clinical practice, we explore the optimal pretreatment methods for different species and morphologies of filamentous fungi. 98 fungal strains were treated with formic acid sandwich method, dispersion method, extraction method, and other methods using a medium element mass spectrometer (EXS3000) as a platform. Each strain had three targets, and the identification rates and confidence differences under different pre-treatment methods were compared to evaluate the identification effects of these methods. The mass spectrometry identification rates of 98 filamentous fungi obtained after pre-treatment with formic acid sandwich method, dispersion method, and extraction method were 85.71%, 82.65%, and 75.51%, respectively. The identification rate of the formic acid sandwich method was significantly higher than the other two methods (P < 0 005) has the best identification ability and the obtained confidence is also higher than the other two methods. The use of formic acid sandwich method for mass spectrometry identification of filamentous fungi can achieve ideal identification results, which is suitable for mass spectrometry identification of filamentous fungi in conventional laboratories.

www.nature.com/scientificreports/identification of filamentous fungi is not yet widely used in microbiology laboratories.There are no standardized guidelines for mass spectrometry pretreatment methods for filamentous fungi, and seeking efficient pretreatment methods is the key to the experiment.The essence of obtaining a spectrum for MALDI-TOF mass spectrometry identification is mainly to obtain the distribution of ribosomal proteins in the strain.The biggest factor affecting its accuracy is the release of bacterial proteins at the target site.Switching between different pre-treatment methods is to obtain the most complete protein types and appropriate protein concentrations of the strain.Due to the different morphologies of different genera of filamentous fungi, the difficulty of obtaining proteins varies greatly when identifying filamentous fungi.Therefore, MALDI-TOF mass spectrometry may have certain differences in identifying different genera of filamentous fungi.In order to seek the optimal pre-treatment method for filamentous fungi, this study will use the Zhongyuan Mass Spectrometer (EXS3000) as a platform to analyze and compare the differences in the identification results of different species of filamentous fungi using three pre-treatment methods: formic acid sandwich method, dispersion method, and extraction method.The aim is to explore the optimal pre-treatment method for filamentous fungi and provide timely pathogenic evidence for clinical practice.

Materials and methods
98 fungal strains were treated using methods such as formic acid sandwich, dispersion, and extraction, with three targets per strain.The accuracy and scores of different types of morphology were compared to evaluate the identification effectiveness of these methods.
Filamentous fungi need to choose an appropriate growth time (generally 3-7 days, for some dark fungi for 10-15 days) to ensure that the fungal body is fresh.When some fungal strains were extracted, the dispersion of fungal bodies was not enough and magnetic beads could be added for grinding extraction.

Source of culture
The comparison strains that have been genetically sequenced provided by Sichuan Provincial People's Hospital have 98 samples, covering several common clinical filamentous fungi such as Aspergillus, Mucor, Penicillium, Fusarium, Cladosporium, Trichosporum and Microsporum bacteria.

Strain culture and identification
Transfer the strain to SDA and incubate it in a mold incubator at 28 ℃ for 2 days.Scotch tape 8 was used to observe the morphological features of hyphae, spores, and spore production structures under a light microscope for identification.For some strains with poor sporulation, the small culture method 9 was used for further identification.

MALDI-TOF MS pretreatment method
Formic acid sandwich method (1) Add 1 μL of formic acid solution dropwise to the target.
(2) Pick a small amount of fungal bodies were taken from the tip of a 10μL straw.
(3) Spread the fungal body on the target in a clockwise direction.(4) Cover with 1 μL of formic acid after drying.
(5) Cover with 1 μL of matrix solution after drying.(6) After drying, it will be tested on the machine.(3) Scrape the freshly cultured fungi from the SDA plate with a sampling swab and rinse them into the extraction solution, repeated several times until the solution is cloudy (4) Vortex and shake to mix well, no need to centrifuge, add 1 μL of suspension solution dropwise to the sample target, and add 1 μL of matrix solution dropwise to cover the sample after drying.

Identification of different pretreatment methods
According to the data, the pre-treatment method using formic acid sandwich method has a good identification effect on filamentous fungi, with an identification rate of 85.71%, followed by the dispersion method with an identification rate of 82.65%.The pre-treatment method using extraction method has a relatively poor identification effect, with an identification rate of 75.51%.See Table 1.

Identification of pretreatment methods for filamentous fungi of different species
According to the results, we can see the pretreatment method with the best identification effect of Aspergillus and Penicillium was formic acid sandwich method, and its identification rates were 94.74% and 83.33%, respectively.The pretreatment method with the best identification effect of Cladospora was the dispersion method, and its identification rate was 88.24%.The identification effect of the three pretreatment methods of Fusarium was good, and the identification rate was 100.00%.The identification results of the three pretreatment methods of Mucormyces and Alternaria were consistent, and the identification rate was 100.00%.See Table 2.

Discuss
Mass spectrometry over flight (MALDI-TOF MS) is a technique for the identification of pathogenic microorganisms at the proteomic level, which has many advantages such as speed, efficiency, and economy.The most difficult thing to identify filamentous fungi by MALDI-TOF MS technique is the pretreatment to extract proteins, which is the main factor affecting the identification results of filamentous fungi.The results of different filamentous fungal protein extraction methods on the identification of mass spectrometry were quite different.Different bacterial www.nature.com/scientificreports/protein extraction protocols directly affect the accuracy of identification 10,11 .The cell wall of filamentous fungi contains more chitin components, and the wall is thick and tough, so it needs to be pretreated to fully rupture the cell wall, and the cytoribosomal protein release can be used for subsequent mass spectrometry analysis.Our study showed that the pretreatment method of formic acid sandwich method had a good identification rate of 85.71% for filamentous fungi, followed by the dispersion method with an identification rate of 82.65%, and the pretreatment method of extraction method had a relatively poor identification effect of 75.51%.The results of the pretreatment methods for different species of filamentous fungi in this study showed that the formic acid sandwich method was the formic acid sandwich method with the best identification effect of Aspergillus and Penicillium, respectively, and the dispersion method was the dispersion method with the best identification effect of C. cladospora, and the identification rate was 88.24%.The identification effect of the three pretreatment methods of Fusarium was good, and the identification rate was 100.00%.The identification results of the three pretreatment methods of Mucormyces and Alternaria were consistent, and the identification rate was 100.00%.This may be less than that of Mucor and Alternaria, which cannot reflect the difference between the three pretreatment methods.. Our study found that the efficiency of protein precipitation was different among different species of filamentous fungi due to the differences in morphology and difficulty of wall breaking, and there were some differences in the identification effect of different pretreatment methods.
Mass spectrometry identification has the highest accuracy, greatly reducing the time required for identification.It can provide timely pathogen diagnosis for clinical practice, help clinical patients choose antifungal drugs correctly, timely control patient infections, and reduce mortality rates.Therefore, considering all factors, it is ideal to culture filamentous fungi in SDA medium for 3 days and extract proteins for mass spectrometry identification using the pre-treatment method of formic acid sandwich.
The limitation of this study is that some filamentous fungi have a relatively low quantity and variety.In future research, we will continue to collect and retain strains, enrich the quantity and variety of filamentous fungi, and include them in the study.Therefore, in subsequent experiments, it is necessary to continue exploring and optimizing various influencing factors for the identification of filamentous fungi by mass spectrometry, in order to better improve the identification rate of filamentous fungi by mass spectrometry.Gradually achieving operational standardization in the identification of filamentous fungi in clinical laboratories 12,13 .It is believed that in the future, MALDI-TOF MS technology will be more and more widely used in the identification of filamentous fungi, and will gradually replace the traditional morphological identification methods.

( 1 )
Add 200 μL of mold extraction reagent I and 200 μL of mold extraction test II to the mold extraction tube.(2) Immerse the sampled swab in the mixed extraction reagent.

Table 1 .
Identification of different pretreatment methods.

Table 2 .
Identification of pretreatment methods for filamentous fungi of different species.